Cilia and flagella, and the basal bodies from which they grow, are highly conserved throughout the evolution of eukaryotic cells. from unicellular protists to various types of differentiated cells in mammalian systems. Basal bodies, which are analogous to centrioles in structure, serve as the organizing site for a complex set of cytoskeletal structures including microtubule rootlets and various striated fibers. These cytoskeletal elements appear to be involved in segregating basal bodies properly during the cell cycle, in determining or maintaining cellular polarity, in positioning of other cellular organelles, and in anchoring cilia and flagella in the cell. The unicellular biflagellate green alga Chlamydomonas has long been used as a model genetic system to study the genes involved in flagellar function. Previous genetic studies of mutants with abnormal flagellar number have shown that this phenotype results from defects in the replication, maturation, and segregation of basal bodies during the cell cycle. The recent development of methods for insertional mutagenesis in Chlamydomonas has greatly facilitated the cloning of genes identified by mutation. These new methods will be used to clone genes involved in basal body function. The specific aims are: 1) to use insertional mutagenesis to identify genes involved in basal body replication, segregation, and localization by isolating mutants with abnormal flagellar number; 2) to clone the genes identified by mutation and to characterize them by DNA sequencing; 3) to examine the function of each gene through detailed cytological analysis of the mutant phenotype; and 4) to examine the spatial and temporal localization of the gene products using immunocytochemistry. It is likely that many of the proteins involved in basal body function identified in this project will have homologs in mammalian cells such as ciliated epithelial cells, sperm cells, and sensory cells. Results from this project will provide a better understanding of the cytoskeleton in these cells.